Chemoenzymatic Hunsdiecker-Type Decarboxylative Bromination of Cinnamic Acids

In this contribution, we report chemoenzymatic bromodecarboxylation (Hunsdiecker-type) of α,ß-unsaturated carboxylic acids. The extraordinarily robust chloroperoxidase from Curvularia inaequalis (CiVCPO) generated hypobromite from H2O2 and bromide, which then spontaneously reacted with a broad range of unsaturated carboxylic acids and yielded the corresponding vinyl bromide products. Selectivity issues arising from the (here undesired) addition of water to the intermediate bromonium ion could be solved by reaction medium engineering. The vinyl bromides so obtained could be used as starting materials for a range of cross-coupling and pericyclic reactions.


General information Materials
All chemicals and reagents were obtained from commercial suppliers (Sigma-Aldrich, Bide Pharmatech Ltd., Alfa Aesar, Macklin, Energy chemical, etc.) and used without further pre-treatments.

Preparation of Vanadium-Chloroperoxidase
The heterologous expression and purification of vanadium-chloroperoxidase from Curvularia inaequalis (CiVCPO) were performed according to our reported procedures 1 .
A 2 L culture of Escherichia coli transformant (E. coli TOP10 (Invitrogen) with the construct pBAD-VCPO) was grown at 37 °C in LB medium supplemented with 100 µg/mL ampicillin to an OD 600 nm between 0.6 and 0.8. Protein expression was induced by adding 0.02 % L arabinose and kept the fermentation broth at 20 °C for 2 hours. The incubation then continued for 24 hours at 25 °C before cell harvest.

Purification of the Enzymes
Cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C (4.3xg). The cells were resuspended to 1 g mL -1 in 50 mM Tris/H2SO4, pH 8.1 fortified with protease inhibitors, lysozyme (2 mg mL -1 ) and DNaseI. Cells were lysed using a cell disruptor and debris was removed by centrifugation at 15000 rpm for 1 h at 4 °C. Then an equal volume of isopropyl alcohol was added to the supernatant to precipitate nucleic acids and unstable proteins. After centrifugation (30 min at 15000 rpm), the clear supernatant was applied to a DEAE Sephacel column (Amersham Pharmacia Biotech) (5mL min -1 ) equilibrated with 50 mM Tris/H2SO4 (pH 8.1). After washing the column with 2 volumes of 50 mM Tris/ H2SO4 (pH 8.1), and 2 volumes of 0.1 M NaCl in 50 mM Tris/H2SO4 (pH 8.1), the enzyme was eluted with 1 M NaCl in 50 mM Tris/HCl. Finally, the pure apoenzyme was dialysed against 100 µM orthovanadate in 50 mM Tris H2SO4, pH 8.1 to obtain the reconstituted holoenzyme. The protein concentration was estimated by the BSA assay.

General procedure for the decarboxylation to synthesise vinyl halides
The α,β-unsaturated carboxylic acid (1a-12a, 30mM), H2O2 (30mM), KBr (50mM), and 400nM CiVCPO were added in a 1mL citrate buffer solution (100mM, pH 5.0). The above concentration represents the final concentration of each ingredient. Then, the mixture was mixed and reacted under the 30°C in a thermal shaker with 800 rpm. The product concentration was obtained by using calibration curves in GC. To determine the selectivity, the integrated peaks of the vinyl product and aldehyde in GC-MS were used.

Semi-preparative synthesis of halogenated ether compounds
In a semi-preparative synthesis (50 mL), the same reaction conditions were applied as described above, except that the amount of the auxiliary solvent (DMSO) was increased to improve the substrate solubility. After reactions, the mixture was extracted with ethyl acetate three times and dried over Na2SO4. The solvent was evaporated at 45 °C and the products were purified by flash chromatography (Biotage rapid preparation system) using 5% -20% ethyl acetate in petroleum ether. The isolated yield is calculated based on the amount of the vinyl halide products in comparison to the substrate added.

Analytics GC analysis
Sample preparation: After the reaction, the reaction mixture was extracted using ethyl acetate containing dodecane as internal standard (5 mM) (extraction ratio: 1:2) and dried over Na2SO4, then, the sample were analysed by gas chromatography (GC). Sample analysis: The GC data were analysed by SHIMADZU GC-2010 Pro equipped with column SH-Rtx-1 (30m × 0.25mm × 0.25µm). The temperature profile was 120 °C holding for 0.8min; 30 °C min -1 to 180°C holding for 1.2 min; 30 °C min -1 to 230°C for 1 min; 30 °C min -1 to 320°C for 0.5 min.

GC-MS analysis
Sample preparation: After the reaction, the reaction mixture was extracted using pure ethyl acetate and dried over Na2SO4. The sample were then analysed by gas chromatography-mass spectroscopy (GC-MS). Sample analysis: Electron ionisation (EI) GC-MS data were collected on an Agilent model 7890A GC with a DB-5 fused silica capillary column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness), Agilent 7200 Q-TOF mass selective detector and 7683B autosampler. The GC was programmed from 60 °C (held for 1 min) to 150 °C at 20°C min -1 (held for 1 min); 20 °C min -1 to 220 °C holding for 1.5 min; 20 °C min -1 to 300 °C holding for 1.5 min; the injection port temperature was 250 °C, and the transfer line temperature was 280 °C using the following parameters: ultra-high purity helium carrier gas, column flow at 1 mL min -1 , injection port temperature 250 °C, transfer line temperature 280 °C. The MS were scanned through full-scan data acquisition from 35 to 550 atomic mass units. 1 H NMR spectra were recorded at 298.2 K on a Bruker AVANCE III 400 MHz NMR spectrometer (Bruker Bio spin, Germany), operating at 400 MHz for proton frequency, and 101 MHz for carbon frequency. TMS was used an internal standard and CD3Cl was used as the solvent.                1 3 4 . 9 1 9 8 . 8 6 2 . 9 9 2 . 0 1 7 0 . 9 1 1 6 . 9 3 7 . 9 2 4 7 . 0 1 5 3 . 0                      Figure S42. Comparison of the selectivity of the conversions of starting materials 1a-12a in the presence of either 5% or 50% DMSO in 1mL scale.

Synthesis of TXT catalyst
The catalyst TXT was synthesized according to previously reported methods [2] .
Step1: The synthesis of bis(3,5-dimethoxyphenyl) sulfane A mixture of iodoarene a (600mg), carbon disulfide (140ul), CuI (0.044g), and DBU (680 ul) in toluene (8mL) were stirred in 25mL three-necked bottle at reflux under N2 for 12 h. The reaction was monitored by using TLC. Upon the completion of the reaction, H2O was added and then the solution was extracted with CH2Cl2. The organic layer was combined and dried over Na2SO4 and concentrated in vacuo. The crude mixture was purified by column chromatography on silica gel (petroleum/ethyl acetate = 85% / 15%) to provide the corresponding bis(3,5-dimethoxyphenyl) sulfane b as a white solid.

Step2: The synthesis of 9-(2-Methylphenyl)-1,3,6,8-tetramethoxythioxanthylium trifluoromethanesulfonate (TXT)
To a solution of thioether b (150mg) and benzoyl chloride (232mg) in chlorobenzene (5.0 mL) was stirred in a 25 mL flask under N2. Then the trifluoromethanesulfonic acid (400ul) was slowly added to the solution and the mixture was heated to 120 °C and kept for 1 h. After cooling to room temperature excess Et2O was added to precipitate the target compound as a solid. The solid was filtered and thoroughly washed with Et2O and dried in vacuo, affording the desired thioxanthylium product d as a brown solid (TXT).

General procedure for the homo-coupling reaction
Agarose (0.05g) and Pd (OAc)2 (0.02mol) were prepared in a water-containing (2 mL) flack (10mL) and heated at 90 o C with stirring for 5 min. Then, the β-bromo styrene (12b) (1mmol) and NaOH (1.5 mmol) were added and stirred at 90 o C under the air for 12 h. After the reaction, the mixture was cooled and the resulting mixture was extracted with acetic ether, and dried under reduced pressure to obtain the corresponding crude product. The residue was purified by flash column chromatography (pure petroleum) providing the desired homo-coupling product.